Methods of reducing the cost required for coming into compliance

ABSTRACT

The present invention relates to apparatus, methods, and applications for treating wastewater, and more particularly to biological processes for removing pollutants from wastewater. This invention further relates to apparatus and methods for growing microbes on-site at a wastewater treatment facility, and for economically inoculating sufficient microbes to solve various treatment problems rapidly

RELATED APPLICATIONS

The present patent document is a continuation-in-part of applicationSer. No. 09/737,718 filed Dec. 15, 2000, which claims the benefit of thefiling date under 35 U.S.C. §119(e) of Provisional U.S. PatentApplication Ser. No. 60/171,264 filed Dec. 16, 1999. All of theforegoing applications are hereby incorporated by reference.

BACKGROUND

In the treatment of wastewater, microorganisms mostly bacteria use thesoluble organic matter in the water as a food source. The bacteriaconsume the organic compounds and convert them to carbon dioxide, water,and energy to produce new cells.

The use of microbes for wastewater treatment and environmental clean upof contaminated soils is well known. Examples of this can be seen inindustry, such as microbial products sold to biological wastewatertreatment plants (WWTP) by United States based companies such asNovozymes of Salem, Va. (Novozymes Biologicals, Inc.); InterBio, Inc. ofThe Woodlands, Tex.; Sybron Corporation of Birmingham, N.J.; or PolybacCorporation of Bethlehem, Pa. These microbial products target variousproblems associated with the operation of the treatment systems.

The basis of these commercial products is the isolation or pre-selectionof microbes from different environments other than the actual site beingtreated. These non-indigenous microbes, hereafter referred to as“exogenous” microbes, are isolated by such companies and grown orfermented under controlled conditions in a manufacturing facility. Fromthe fermenter, the pure culture of microbes is concentrated into apaste, reconstituted, and placed on an inert carrier, such as bran,oatmeal, rye, or cornhusks. These carrier materials are often sterilizedto reduce the natural background contamination with other undesirablefecal organisms or unwanted microbes. These unwanted or non-targetmicrobes can become a significant part of the final product. Thisreconstituted mixture then undergoes a stabilization procedure, usuallyfreeze-drying. Even with the use of cryo-protectants in thereconstituted mixture to protect the microbes, this process generallykills in excess of 90% of the microbes. Therefore only about 1-10% ofthe microbes can be recovered after freeze-drying. Air-drying, a postliquid fermentation process, is also used by some companies to stabilizethe microbes, but still results in high losses and poor recovery ofviable microbes. After stabilization, different microbes are blendedinto formulations to address different operational problems or tobiodegrade various environmental pollutants.

The process of adding these exogenous microbes to a biologicalwastewater treatment system is termed “bioaugmentation,” because it isadding or enhancing the existing biological fauna. Using currenttechnologies, the application of exogenous microbes often has no effector insufficient impact, resulting in the plant violating NationalPollutant Discharge Elimination System (NPDES) permits issued by theEnvironmental Protection Agency (EPA), or violating other environmentalregulations, including state or local environmental regulations, andenvironmental regulations of countries other than the United States.Currently, the typical reaction time is 2-3 weeks for bioaugmentation totake effect. Since the NPDES Permits are monthly, this only leaves abouttwo weeks or less to identify that the NPDES Permit is jeopardized,which is insufficient time for the plant to address the problem.

There is a need to be able to quickly, reliably, and economicallycontrol biological wastewater treatment plant upsets in order todecrease the levels of contaminants in wastewater and to avoid violationof NPDES Permits and other environmental regulations regarding pollutantdischarge.

BRIEF SUMMARY

The present invention relates to apparatus, methods, and applicationsfor treating wastewater, and more particularly to biological processesfor removing pollutants from wastewater. This invention further relatesto apparatus and methods for growing microbes on-site at a wastewatertreatment facility, and for economically inoculating sufficient microbesto solve various treatment problems rapidly. The system may be appliedto growing microbes on-site for clean up of contaminated soils orgroundwater treatment. The system may also be modified to become aspecialized treatment system for biodegradation of liquid hazardouswastes on-site, eliminating the need for hauling away hazardous wastesfor remote disposal.

The fermentation system of the present invention provides numerousimprovements in bioaugmentation systems by increasing the efficiency ofwastewater treatment. In accordance with the present invention, afermentation system for providing microbes to degrade waste organiccompounds present in a water mixture comprises a fermentation tankprovided with aeration, mixing, and maintained within a giventemperature range. Further, in accordance with the present invention, acarbon source, nutrients and selected microbes are provided.

In one aspect of the present invention, the fermentation system ison-site at the waste water treatment plant (WWTP), thereby reducing highshipping costs of transporting the inoculum to the WWTP.

In another aspect of the present invention, the effective concentrationof the desired or target microbes in the inoculum with which thewastewater is treated is increased, thereby increasing the efficacy andefficiency, and thereby reducing the per unit cost of treatment.

In another aspect of the present invention, isolation of the indigenousfunctional, desired, or target microbes and fermentation on-site,outside the competitive environment of the WWTP, enhances the efficacyand effectiveness of such applications, since the indigenous populationtends to have more stable genetic characteristics.

In another aspect of the present invention, the proportion of target,functional microbes in the inoculum is increased, and the proportion ofnon-target, non-functional microbes is decreased, adding to the efficacyand efficiency of dosing at the point of application.

In another aspect of the present invention, the inoculum is fed into thefermentation tank through an automated process.

In another aspect of the present invention, various parameters of thefermentation system are monitored and controlled, such as pH,temperature, nutrients, carbon sources, aeration, and mixing.

In another aspect of the present invention, various parameters of thefermentation system are monitored and controlled through an automatedprocess.

In another aspect of the present invention, various parameters of thefermentation system are monitored and controlled from a remote location.

In another aspect of the present invention, there is provided a methodof removing contaminants from an aqueous liquid comprising depositing aninoculum comprising microbes into a fermentation system; fermenting theinoculum in the fermentation system to provide a treatment batchcomprising the microbes; and applying at least a portion of thetreatment batch to an aqueous liquid.

In another aspect of the present invention, there is provided a methodof removing contaminants from an aqueous liquid, comprising depositingan inoculum comprising microbes into a fermentation system in aconcentration of 10³ cfu/ml to 10⁸ cfu/ml; fermenting the inoculum inthe fermentation system to provide a treatment batch comprising themicrobes in a concentration of 10⁶ cfu/ml to 10¹⁰ cfu/ml; and applyingat least a portion of the treatment batch to an aqueous liquid toprovide a microbe concentration in the aqueous liquid of at least 10³cfu/ml.

In another aspect of the present invention, there is provided a methodof removing contaminants from an aqueous liquid, comprising: depositingan inoculum comprising microbes into a fermentation system; addingnutrient, water and defoamer to the fermentation system to provide afermentation mixture; fermenting the fermentation mixture to provide atreatment batch comprising the microbes; and applying at least a portionof the treatment batch to an aqueous liquid. The fermenting may compriseheating, mixing and aerating the mixture in the fermentation system.

In another aspect of the present invention, there is provided a methodof removing contaminants from wastewater in a biological wastewatersystem, comprising depositing an inoculum comprising microbes into afermentation system in a concentration of 10³ cfu/ml to 10⁸ cfu/ml;fermenting the inoculum in the fermentation system to provide atreatment batch comprising the microbes in a concentration of 10⁶ cfu/mlto 10¹° cfu/ml; and releasing at least a portion of the treatment batchdirectly into the wastewater to provide a microbe concentration in thewastewater of at least 10³ cfu/ml.

In another aspect of the present invention, there is provided a methodof removing organic contaminants from a wet well, comprising depositingan inoculum comprising microbes into a fermentation system; addingnutrient, water and defoamer to the fermentation system to provide afermentation mixture; fermenting the fermentation mixture to provide atreatment batch comprising the microbes, the fermenting comprisingheating, mixing and aerating the mixture in the fermentation system; andapplying at least a portion of the treatment batch to a wet well. Theorganic contaminants may comprise fat, oil, grease, or mixtures thereof.

In another aspect of the present invention, there is provided a methodof removing contaminants from soil, comprising depositing an inoculumcomprising microbes into a fermentation system; adding nutrient, waterand defoamer to the fermentation system to provide a fermentationmixture; fermenting the fermentation mixture to provide a treatmentbatch comprising the microbes; and applying at least a portion of thetreatment batch to a soil surface. The fermenting may comprise heating,mixing and aerating the mixture in the fermentation system

In another aspect of the present invention, there is provided a methodof reducing the time required for coming into compliance with anenvironmental discharge regulation comprising fermenting an inoculumcomprising microbes to provide a treatment batch; and administering atleast a portion of the treatment batch to a wastewater treatment system.

In another aspect of the present invention, there is provided a methodof reducing the cost required for coming into compliance with anenvironmental discharge regulation comprising fermenting an inoculumcomprising microbes to provide a treatment batch; and administering atleast a portion of the treatment batch to a wastewater treatment system.The cost required for coming into compliance with the environmentaldischarge regulation may be reduced by at least about 25 percent.

In another aspect of the present invention, there is provided a methodof reducing the amount of settling aid required by a wastewatertreatment system comprising fermenting an inoculum comprising microbesto provide a treatment batch; and administering at least a portion ofthe treatment batch to a wastewater treatment system. The amount ofsettling aid used in the wastewater treatment system may be reduced byat least about 25 percent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Conceptual Diagram of Fermentation Process

FIG. 2 Multi-Stage Fermentation Process Flow Diagram

FIG. 3 Modified Multi-Stage Fermentation Process To Hazardous WasteTreatment Process Flow Diagram

FIG. 4 Diagram of Spray Bar

FIG. 5 Schematic Diagram of Automated Fermentation System

FIG. 6 Schematic Diagram Multi-stage Fermentation System

FIG. 7 Schematic Diagram of One Embodiment of Manual Fermentation System

FIG. 8 Schematic Diagram of One Embodiment Automated Fermentation System

DETAILED DESCRIPTION

The apparatus and methods of the present invention provide a quick,reliable, and economical way of controlling biological wastewatertreatment plant upsets in order to avoid violation of environmentaldischarge regulations such as NPDES Permits, other discharge permitrequirements, or other environmental regulations regarding limits oneffluent materials that can be introduced into the environment. Suchregulations vary from state to state, and from country to country. Theterms “environmental discharge regulation” and “discharge permit” areused to describe such permits and regulations including, but not limitedto, NPDES Permits, other discharge permit requirements, or otherenvironmental regulations regarding limits on effluent materials thatcan be introduced into the environment. Optimally, this involves anon-site fermentation process, which can increase the effective dosage ofmicrobes by 10-100 fold or more over the existing technology. In oneembodiment, the fermentation system is an on-site, automated, continuousfermentation system with the features of deliberate aeration, heating,automated sterilization, automated feeding of raw materials, automatedinoculation, with multi-stage fermentation vessels or multiple dailycycles along with remote monitoring capability of the process forquality assurance and quality control purposes. In addition, the systemmay be weatherproof for on-site application and capable of automatedinoculation and re-inoculation from one batch to the next.

This apparatus may be applied to a range of aerobic, facultative, oranaerobic biological wastewater systems. Aerobic processes include, forexample, activated sludge systems, aerobic stabilization basins (ASB),aerated lagoons, single pass lagoon systems, stabilization ponds,rotating biological contactors, and trickling filters. Facultativeprocesses include, for example, facultative lagoons. Anaerobic processesinclude, for example, anaerobic ponds, anaerobic digesters, anaerobicfilters or contactors, and anaerobic treatment systems. Differentmicrobes are grown to address different environmental or operationalproblems, including psychrophilic, mesophilic, or thermophilicheterotrophs for biodegrading organic pollutants or lithotrophs, such asnitrifying bacteria (Nitrosomonas and/or Nitrobacter) for nitrification.For anaerobic processes, microbes such as methanogens, methylotrophs orother anaerobic synthrophic microbes may be grown. Names of usefulanaerobic microbes can be found in “Filamentous and Dispersed Growth inAnaerobic Contact Systems, G. R. Whiteman, Ph.D. Thesis, 1985 Universityof Newcastle-upon-Tyne, England or Advanced Biological Services (ABS)Inc., Duluth, Ga. USA, which is incorporated herein by reference.

The use of the functional, target, or desired indigenous microbes(referred to as target indigenous microbes) isolated from the biologicalWWTP for growth in such an apparatus enhances its effectiveness furtherby dosing high numbers of active, acclimated microbes. The process ofusing such indigenous microbes has been termed Natural Bioaugmentation.

One aspect of the present invention is the sufficient, repeatedinoculation of the functional microbes (whether an exogenous orindigenous source) that allows a microbial population to be establishedquickly and out-compete an undesirable indigenous population, such asfilamentous or Zoogloeal type microbes, which cause bulking. There is atremendous commercial need to solve such problems as filamentous orZoogloeal bulking, which can increase the total costs of operating awastewater treatment plant by as much as 20-25%. The first area ofincreased treatment costs arise because of the need to use settling aidsor chemicals to clarify the water and concentrate the biomass in thesecondary clarifier. Examples of such chemicals include polymer,bentonite, alum, or ferric salts. The second area of increased costsarise due to the poor dewaterability of filamentous and Zoogloealgrowth, thereby increasing the use of polymer for dewatering, and hencethe costs. The fermentation system of the present invention provides aprocess which is applied to control or displace undesirable microbes,such as filamentous or Zoogloeal type microbes which cause bulking andsettleability problems. The fermentation process is used to decrease oreliminate the use of polymers to enhance settling, to minimize the useof dewatering chemicals, and to minimize the need for sludge handlingand disposal.

In accordance with one embodiment of the present invention, afermentation apparatus comprises a fermentation tank provided with a gastransfer system and mixing, and maintained within a given temperaturerange. The gas transfer system may provide aeration when growing aerobicmicrobes, or recirculation of anaerobic gases, such as methane,hydrogen, and carbon dioxide, when growing anaerobic microbes. Theprocess of gas transfer may provide sufficient mixing, or mixing may beprovided by a separate mechanism. The desired temperature range may bemaintained through the use of a heating source. Alternatively, thedesired temperature of the fermentation tank may be maintained bylocating the tank in an area with the appropriate ambient temperature.For example, the tank may be located in a factory or mill in proximityto some type of equipment which generates enough heat to heat the tankto the desired temperature.

Fermentation nutrients such as carbon sources and additional nutrientsare also provided into the fermentation tank. These nutrients may beprovided manually, or through the use of an automated system which feedsthe fermentation nutrients directly into the fermentation tank.

One aspect of the present invention is to provide an effectiveconcentration of desired microbes at a point of application sufficientto significantly treat the wastewater at the application point.Optimally, the inoculum is grown to a concentration of approximately10⁹-10¹⁰ colony forming units per milliliter (cfu/ml) to achieve apreferred minimum inoculation of approximately 10⁴ cfu/ml at the pointof application.

The foregoing is illustrated by the following example of an industrywith a 40 million gallons per day (MGD) flow of wastewater. Aninoculation of approximately 10⁴ cfu/ml is provided to the 40 MGD flowof wastewater by using a 400 gallon tank of inoculum grown to 10¹⁰cfu/ml, a 4000 gallon tank of inoculum grown to 10⁹ cfu/ml, or a 40,000gallon tank of inoculum grown to 10⁸ cfu/ml.

Clearly, the volume of inoculum required, the effective concentration ofthe inoculum required, and the volume of wastewater to be treated areall interdependent. Further, the optimal numbers provided here, of theinoculum being grown to a concentration of approximately 10⁹-10¹⁰cfu/ml, and of achieving approximately 10⁴ cfu/ml at the point ofapplication, are based on organisms currently used and commonly known.One of ordinary skill in the art can determine what effective dosage ofmicrobes is best, and as microbes are discovered that are more efficientin degrading contaminants, lower levels of those microbes may berequired.

In one embodiment, microbes are provided for treatment of variouswastewater problems in the following manner. An inoculum of microbes isprovided into a fermentation tank. Fermentation nutrients required formicrobial growth, such as carbon sources, macro- and micro-nutrients,selective carbon sources, and selective nutrients are also supplied.Preferably, air and mixing are also supplied. The inoculum is grown inthe fermentation tank for a sufficient number of generations untilreaching the desired concentration of cfu/ml. Then the inoculum isprovided at the point of application for wastewater treatment.

The types of microbe or microbes present in the inoculum depend on thetype of wastewater to be treated. The inoculum may contain a singlestrain or multiple strains of microbes depending on the wastewaterproblem being addressed. Inoculum may be provided as a liquid or a dryproduct. Dry product is commonly freeze dried or air dried.Additionally, the microbes may be exogenous to the wastewater orindigenous microbes may be isolated from the wastewater under treatment.

The fermentation process can be carried out in multiple tanks or in asingle multi-compartmentalized tank. One advantage of using multipletanks is to reduce the scale at the front end of the process, thereby,reducing the amount of inoculum which must be stored, reducing the cost,and increasing the efficiency.

For example, if a final concentration of 10⁹ cfu/ml is desired, afermentation tank can be inoculated with a 10⁹ cfu/ml mother culture ina volume one-hundredth that of the fermentation tank. The term motherculture is used to refer to the desired, functional or target microberequired to achieve the treatment goals, or the original culture ofmicrobes used for inoculation. The source of the microbes in the motherculture may be exogenous or indigenous microbes. This yields a startingconcentration in the fermentation tank of 10⁷ cfu/ml. To grow from 10⁷cfu/ml to 10⁹ cfu/ml, the culture must grow 100 fold, or approximatelyseven generation times. If, for example, the doubling time for a givenbacterial strain is one-half hour, it would take approximately three andone-half hours to grow from 10⁷ cfu/ml to 10⁹ cfu/ml, at whichconcentration, the batch would be ready to release at the point ofapplication.

The inoculum provided at the start of a fermentation batch is optimallyabout 10⁶-10⁷ cfu/ml, and may be in the range of about 10³ to 10⁸cfu/ml. By inoculating with a concentration of microbes at about 10⁷cfu/ml, it is likely that the target microbe of the inoculum would beable to overwhelm any background contamination that may be present.

In accordance with the present invention, it is possible to multicyclethe growth of microbes on a daily basis. In other words, depending onthe generation time of the microbe in question, it may be possible togrow more than one batch of inoculum in a single day. The variousbatches may comprise the same or a different microbe or group ofmicrobes. The time required for the growth of a batch of microorganismsdepends on the doubling time, or generation time, for the organism. Theoptimal concentration of microbes also depends on the type of microbeused, and how efficient the microbe degrades the organic in question.What is required is an “effective dosage of organisms” to enhance theefficacy of the treatment.

It is well known that Gram negative microbes such as Pseudomonas speciesare the workhorses of most industrial biological treatment systems. (TheGram stain is a basic criteria used to categorize groups of bacteria, aseither Gram positive or Gram negative, based on differential staining ofthe bacterial cell wall.) Occasionally, Gram positive microbes willdominate waste streams containing large amounts of carbohydratematerials, such as molasses or starch.

The industry competitive standard for exogenous microbial products is10⁹ colony-forming units per gram (cfu/g). Generally, off-the-shelfcommercially available exogenous microbial products contain about 1-10%target, deliberately added Gram negative microbes, such as aPseudomonas. This means that the viable counts for the non-fecal, Gramnegative microbes only range from 10⁷ to 10⁸ cfu/g.

These microbial products are generally filled with easy-to-grow,“non-functional” or “non-target” microbes, such as the Gram positiveBacillus, rather than Gram negative microbes, such as Pseudomonasspecies, in order to meet the industry competitive standard of 10⁹colony-forming units per gram (cfu/g). Bacillus species are a Grampositive rod-shaped microbe, which form spores when exposed to non-idealconditions. These microbial spores are the inert, non-active form of themicrobe. Generally, any upset biological treatment plant has non-idealconditions to which the indigenous microbial fauna have been exposed andgenerally continue to be exposed, which is why the natural biologyrecovers slowly. For these reasons, any Bacillus species will tend toform spores when introduced into the treatment system, and, therefore,will provide no beneficial effect to enhancing the biology of theprocess.

The microbes found in biological treatment systems often carryextraneous genetic information in the form of plasmids for biodegradingenvironmental pollutants, such as toluene, xylene, naphthalene, andother organics. In the process of manufacturing the microbes, reversionor loss of these plasmids is common, since there is no selectivepressure for the microbe to maintain the plasmid. In other words, thisextraneous genetic information is not essential to growth andreproduction of the microbe under the ideal conditions found in aconventional manufacturing facility. Loss of the plasmid encoding theinformation for toluene biodegradation, for example, makes the microbeineffective in treating that pollutant.

The type of microbe or microbes, which will be most effective for aparticular application will depend upon the application, the wastewatercharacteristics, and the characteristics that are required to solve theparticular problem. For example, microbes may be selected based on theirability to degrade pollutants on the list of Federal Prioritypollutants, which is incorporated herein by reference.

The terms microorganism, microbe, or organism, as used herein, areinterchangeable and, include fungus, yeast, bacteria, and otherbiodegrading small unicellular organisms. Some examples of microbes withparticular biodegradation characteristics are provided in Table 1.

TABLE 1 Respiration Application Microbe Type Example End Product ExamplePseudomonas putida Aerobic Phenol, toluene Water, CO₂, biomass Bacillussubtilis Aerobic Starch Water, CO₂, biomass Nocarida spp. AerobicCyclohexane Water, CO₂, biomass White Rot Fungus spp. AerobicChloro-organics Water, CO₂, biomass Nitrosomonas spp. Aerobic Ammoniaoxidation Nitrite Nitrobacter spp. Aerobic Nitrite oxidation NitrateThiosphera pantotropha Aerobic Denitrification Nitrogen gas Methanogenicbacteria Anaerobic Acetic acid Methane, CO₂, biomass Notes: Spp. =species, these can vary; Subtilis is one species of Bacillus; Putida isone species of Pseudomonas; CO₂ = carbon dioxide.

Examples of microbes such as Pseudomonas putida and Nocardia strains forthe biodegradation of hydrocarbons are well documented in “Developmentsin Biodegradation of Hydrocarbons-1” by Watkinson, Applied SciencePublishers, 1978 ISBN: 0-85334-751-4, which is incorporated herein byreference. Chloro-organics biodegradation using White Rot Fungus is welldocumented in U.S. Pat. No. 4,554,075, which is incorporated herein byreference. Culture methods are discussed in “Increasing LigninolyticEnzyme Activities in Several White-Rot Basidiomycetes by NitrogenSufficient Media” Erwin et al., Biosource Technology, Volume 53, (1995),pages 133-139, Elsevier Science Limited, which is incorporated herein byreference. The biodegradation activities of the other microbes listedabove are all well documented in Bergey's “Manual of SystematicBacteriology”, along with many other useful microbes. (Bergey's “Manualof Systematic Bacteriology” Williams & Wilkins, London, 1984 ISBN:0-683-04108-8, four volumes in total published every 4 years, which isincorporated herein by reference). Any of the Culture Collectionsprovide biodegradation characteristics for various cultures on file.Examples of catalogues of microbes that can be obtained include theAmerican Type Culture Collection—“Catalogue of Bacteria andBacteriophages”, 17^(th) edition, 1989, ISBN: 0-930009-23-1, and the“Catalogues of the National Collections of Type Cultures and PathogenicFungi”, The Public Health Laboratory Service (PHLS), 1989, ISBN0-901144-25-8, the disclosures of which are incorporated herein byreference.

Off-the-shelf products can be bought from USA Manufacturers previouslymentioned such as Novozymes, Polybac Corporation, InterBio Inc., orSybron Corporation. When treating wastewater for phenol contamination,for example, one could buy Phenobac from Polybac Corporation, or CX80from InterBio Inc., or Bichem 1002 CG from Novozymes or SybronCorporation. All these products are claimed, by the manufacturer, tobiodegrade phenol.

Standard microbiological isolation methods described in “IdentificationMethods in Applied and Environmental Microbiology” by Board et al. canbe used for isolation and identification of indigenous microbes fromsamples obtained from the site. (“Identification Methods in Applied andEnvironmental Microbiology”, R. G. Board, Dorothy Jones, and F. A.Skinner, Blackwell Scientific Publications, London, 1992 “The Societyfor Applied Bacteriology Technical Series No. 29,” ISBN: 0-632-03379-7,the disclosure of which is incorporated herein by reference.).

Many suitable media can be obtained commercially from Oxoid ofHampshire, England, or Difco of Detroit, Mich., distributors of mediaincluding non-selective media such as plate count agar (PCA).Alternatively, selective media can be used such as Psuedomonas Isolationagar (PIA), which will isolate just Pseudomonas. Other sources ofinformation on special culture media for isolation and identification ofspecific microbes includes the “Handbook of Microbiological Media,”Ronald M. Atlas, CRC Press, edited by Lawrence C. Parks, ISBN:0-8493-2944-2; and Bergey's “Manual of Systematic Bacteriology” Williams& Wilkins, London, 1984, the disclosures of which are incorporatedherein by reference.

Once isolated in pure culture, identification of the microbes can beachieved through characterization procedures described in the abovereferences. Alternatively, such isolates can be sent for identificationto experts at the American Type Culture Collection (ATCC) or NationalCollection of Type Cultures (NCTC), where bacteria, fungi and other lifeforms are maintained. These Culture Collections are also an alternativesource of cultures to commercially available products from U.S.companies such as Novozymes of Salem, Va.; Polybac of Bethlehem, Pa.;InterBio of The Woodland, Tex.; Sybron of Birmingham, N.J.; or othermanufacturers of biological products. One advantage of sourcing microbesfrom a manufacturer is that microbes can be obtained as single culturesor blended into specific formulations for different applications, suchas oil degradation or treatment of pulp and papermill wastewater.

Determining which culture or manufacturer's formulation is the mosteffective for treating a particular wastewater can be done usingstandard respirometric techniques. The principle of repirometry is tomeasure the activity of a biomass exposed to a test substrate comparedto a control containing biomass and a known substrate which gives apredictable result. The substrate to be tested can range from a specificchemical or waste stream to a combined wastewater. Respirometryexperiments can be set up to stimulate either aerobic or anaerobicenvironments. Typical applications of respirometry include assessing thefollowing: treatability of municipal and industrial wastewater; toxicityof specific waste streams or chemicals; biodegradability of chemicals;biochemical oxygen demand (BOD); and oxygen uptake rates (OUR).

Aerobic microorganisms use oxygen to grow and to metabolize organicsubstrates. For aerobic microbes, oxygen uptake rate (OUR) is consideredto be directly related to organic stabilization, and hence, related tothe ability of the formulation to biodegrade the organic waste.

Respirometry equipment and treatability procedures for both aerobic andanaerobic studies can be obtained from manufacturers in the U.S. such asChallenge Environmental Systems of Fayetteville, Ark.; Arthur Technologyof Fond du Lac, Wis.; and Bioscience Management of Bethlehem, Pa.Examples of aerobic treatability studies can be found in technicalpapers such as Whiteman, G. R., TAPPI Environmental Conference—“TheApplication of Selected Microbial Formulations in the Pulp and PaperIndustry,” TAPPI Environmental Proceedings, Book 1, pp. 235-238, April1991; Whiteman, G. R., Gwinnett Industrial Conference—“OptimizingBiological Processes—A Look Inside The Black Box,” April 1995; andWhiteman, G. R., TAPPI Environmental Conference—“Improving TreatmentPerformance with Natural Bioaugmentation”, TAPPI EnvironmentalProceedings, Vancouver, BC, 1998; the disclosures of which are herebyincorporated by reference.

Once the effectiveness of each isolate, isolates, and/or formulationhave been compared using respirometry techniques, then the best can beselected as the inoculum for the fermentation process described herein.

Nutrients include both macro-nutrients and micro-nutrients. The typicalcomposition of a microbe is shown below in Table 2, in which it isapparent that different microbes have different compositions. Microbesalso have different abilities to assimilate nitrogen into amino acids,the basic building blocks of proteins or the purine or pyrimidine basesof ribonucleic acid (RNA) and deoxynucliec acid (DNA). Therefore,different microbes have different requirements for macronutrients(nitrogen and phosphorus) and micronutrients (for example magnesium,calcium, potassium, sodium, manganese, cobalt, nickel, zinc, iron,chloride and sulfur) to optimize the fermentation process. SeeIntroductory Microbiology by Levy, et al., the disclosure of which isincorporated herein by reference, for information on macronutrients andmicronutrients including concentrations for fastidious (hard to grow)microbes, how to determine whether a particular micronutrient isrequired, and explanations of the role of nutrients in general.

TABLE 2 CONTENT YEAST BACTERIA ZOOGLOEA Carbon (C) 47.0 47.7 44.9Hydrogen 6.0 5.7 — Oxygen 32.5 27.0 — Nitrogen (N) 8.5 11.3  9.9 Ash 6.08.3 — Empirical formula C₁₃H₂₀N₂O₇ C₅H₇NO — C:N ratio 5.6:1 4.3:1 4.5:1

Active biomass, mainly composed of bacteria, in a biological treatmentplant contains 8-15% nitrogen for most bacteria, most typically12-12.5%, and 2-5% phosphorus, most typically 2.3-2.6%. Phosphorus isimportant in the formation of adenosine triphosphate (ATP) which is howmicrobes store energy.

Microbes are composed of proteins, carbohydrates, fatty materials calledlipids, or combinations of these substances. In particular, the proteinsare used to make enzymes that are the basis of the biodegradationprocess. A series of reactions makes up the biodegradation process forany particular organic substance. A specific enzyme carries out eachreaction. These enzymes are composed of amino acids and sometimesco-factors, usually metals, which make up the reactive sites of theenzymes where the biodegradation and conversion of an organic substancetakes place. Optimally, micro-nutrients are present in sufficientquantity in order to optimize the fermentation process. Micro-nutrientsinclude substances such as vitamins, co-enzymes, metals, or inorganiccompounds required such as cofactors for the production of enzymes,coenzymes or for cell growth. For example, sulfur is required for theassimilation of the essential amino acids cystiene and methionine.Information regarding the role of such micronutrients such as coenzymes,including folic acid, pantothenic acid (Coenzyme A), vitamin B₁₂(cobamide), biotin, nicotinic acid or nicotinamide (NAD), vitamin B₁(thiamin), vitamin B₂ (riboflavin), vitamin B₆ (pyroxidine), lipoic acidand ascorbic acid is found in Biochemistry, Second Edition, Albert L.Lehninger, Worth Publishers Inc., 1975, ISBN: 0-87901-047-9, andIntroductory Microbiology by Levy, et al., John Wiley & Sons Inc., 1973,ISBN 0-471-53155-3, the disclosures of which are incorporated herein byreference.

As mentioned earlier, the type of microbe or microbes used in thewastewater treatment process of the present invention depends on thetype of wastewater problem to be addressed. The microbes used most oftenare bacteria, and most commonly, aerobic, mesophilic bacteria are used.Aerobic bacteria use oxygen to metabolize organic matter, as measured,for example, by biochemical oxygen demand (BOD), chemical oxygen demand(COD), total organic carbon (TOC), or total carbon (TC). It is alsopossible to use facultative bacteria, which can metabolize with orwithout oxygen, or anaerobic bacteria, which do not use oxygen. Bacteriaare also classified with respect to the temperature at which they growoptimally. Optimum temperatures are, for thermophiles—55-75° C.; formesophiles—30-45° C.; and for psychrophiles: obligate—15-18° C.

An example of growing a mesophilic microbe is provided as follows: Thegrowth of an aerobic, mesophilic Psuedomonas in the fermentation systemis maintain at the temperature of 35° C. +/−1° C., with residualdissolved oxygen concentrations of greater than 2 mg/l and pH 7.5+/−0.2units. A typical example of a selective nutrient supplement forPseudomonas putida would be phenol, which assists in selecting andfavoring the growth of the Pseudomonas which has phenol degradationcapability. Thus Pseudomonas putida could outgrow any backgroundcontamination and reversion is minimized. Reversion being where theplasmid coding for phenol degradation is lost by the microbe due to acompetitive disadvantage by retaining unnecessary genetic material. Atypical example of a selective inhibitor for Pseudomonas is PseudomonasIsolation Broth (PIB), which contains the chemical called cetrimide thatselectively inhibits the growth of other microbes, and, therefore,favors the growth of Pseudomonas, hence the term selective inhibitor.PIB also contains Tryptic Soya broth and various micronutrients. Suchselective agents may be mixed with the main carbon source, along withmacronutrients and micronutrients and other materials, such as defoamer.The main carbon source generally including concentrated simple sugarssuch as glucose, or molasses, or material used in the manufacturingprocess, such as black liquor at a Kraft mill, starch at a papermill orfood plant, or beer or molasses at a brewery. At a chemical plant themain carbon source may also be a selective nutrient or inhibitor, suchas phenol. The carbon is as concentrated as possible with a BODgenerally of at least 5000 mg/L and typically in the region of50,000-100,000 mg/l in order to reduce raw material volumes required.Nitrogen is provided as ammonia-nitrogen or urea and phosphorus asphosphoric acid both of which are generally used at industrialwastewater treatment facilities to supplement the macronutrientsnitrogen and phosphorus. Micronutrients are occasionally required whereraw water supplies or the selective supplement nutrient or nutrient doesnot contain sufficient minerals to optimize growth.

Defoamer is sometimes be used to prevent foam build-up in the tank,which can occur in vigorous biological fermentation processes.Preferably, such defoamer is water-based or silicone based in naturewith oil-based being avoided. These are readily commercially availablefrom U.S. companies such as Callaway of Columbus, Ga.; Hercules ofWilmington, Del.; Nalco of Naperville, Ill.; and Ashland of Columbus,Ohio. Defoamer is also available at industrial sites where it isroutinely applied for foam control of the biological treatment processesor to prevent foam formation on the river at the point of discharge.

The fermentation of the Pseudomonas is optimized at the site for theparticular raw materials used in the fermentation process.

There are many characteristics of the microbial growth, which may bemonitored and controlled to maintain optimal growth conditions. Thesegrowth parameters include pH, temperature, oxygen levels, conductivity,turbidity and nutrients, such as ammonia and phosphate or micronutrientssuch as iron or sulfur.

Optimal growth parameters are defined based on the type of microbe beinggrown. Using those parameters, growth conditions are set and controlled.The monitoring and control of growth parameters is carried out manually,or through an automated system (24, FIG. 1), or a remotely controlledautomated system. The automated monitoring system optimally has thecapacity to download data regarding the fermentation system and itsparameters. For example, data on the growth parameters which aremonitored during the fermentation process by on-site sensors may bedownloaded at a remote location to provide the operator informationabout the fermentation process.

For the automated system, redundant mechanisms are preferably put inplace such that the failure of a given component does not halt thefermentation process, but results in the activation of the back-upmechanism. For example, if a given pump or power source should fail,there is an automated mechanism that activates the back-up pump orback-up power, respectively. Optimally, the back-up power system islinked to a memory device so information about the stage at which powerwas lost is retained. In this way, when power is regained, thefermentation cycle is initiated at the correct stage of the cycle, andthe fermentation cycle continues from the stage at which power was lost.

The back-up system of the automated control system is optimally equippedwith a paging system or an alarm system by which an operator in a remotelocation is notified of a power failure or other equipment failure. Froma remote location, the operator can receive notification of the failureand the activated back-up systems, and can respond accordingly.

The automated monitoring and control system eliminates the need forsomeone to be on-site to monitor and control the process. Through theuse mechanisms such as remote downloading of data and an automatic pagersystem, an operator can manage a fermentation site or sites from aremote location. This also results in a decrease in the cost to run sucha fermentation system.

When microbes are growing at a rapid rate, the pH can become veryacidic. To maintain optimal growth conditions, the pH is neutralized.Rapid growth rates can also result in temperature variations. Real timecontrol of temperature and pH are particularly desirable.

Oxygen consumption, as measured by oxygen uptake rate (OUR), is directlyrelated to the rate at which the microbes are growing and degradingorganics. Typical OURs for growing cultures may be about 20 mg/l/hr ofoxygen, although it is possible to reach rates of 100-300 mg/l/hr ofoxygen for rapidly growing cultures. It is desirable to provide aerationsufficient to supply oxygen to replace the amount being consumed. Airfilters are preferably placed on the incoming air supply. The filterstypically have a pore size of approximately 0.2 microns or less, andcharged filters may be used. The pore size of the filter is sufficientlysmall to substantially reduce or eliminate contamination with microbesand particles in the air supply.

If an anaerobic bacteria is grown, the air in the fermentation tank isremoved by recycling anaerobic gases and maintaining a closedpressurized system to keep air out.

The monitoring and control of various growth parameters during thefermentation process may occur locally with an option to access andcontrol the fermentation process from a remote location. The controlsystems may be located in the same building or at a greater distancefrom the point of fermentation. In one aspect of the current invention,the growth of microbes is monitored during the fermentation by sensorsmeasuring changes in turbidity, conductivity, temperature, pH, and/oroxygen consumption, and additional nutrients, carbon sources, selectivecarbon sources, oxygen levels, temperature, and/or pH are automaticallyadjusted to optimize growth conditions. For example, pH can becontrolled by the addition of acidic or caustic materials; oxygen levelscan be controlled by the addition or removal of oxygen; and nutrientsand/or carbon sources can be added.

The fermentation tank is preferably washed between different batches ofmicrobial growth to remove attached growth at the waterline, which maycause build-up of contamination. In one embodiment, the tank is washedusing a spray bar mechanism 30, as illustrated in FIG. 4. The spray bar30 is used to introduce washing or sterilization liquids, defoamer, orother liquids into the fermentation tank 32. In one embodiment, a highpressure water supply enters at the end or in middle of the spray bar34. Water can be used from a main water line or recirculated by a highpressure pump which can also be used to transfer the bulk fluid to thepoint of application or the next tank in the series. The spray bar 34can be made, for example, of ¾ inch UPVC (unplasticized poly vinylchloride). Spray heads or sprinkler heads 36 are preferably placed suchthat the spray from the sprinkler heads 36 overlaps. The outer spraybars 38 is preferably located about 5″ above the water line and angleddown in order to achieve 30-60 psi on contact. This degree of pressureis preferred to dislodge any contamination build up at the water level.Another smaller spray bar system 40 in the center ensures the roof ofthe fermentation system is washed.

The tank may be cleaned with water only or by using a small amount ofsurfactant to assist in dislodging material off the surface of the tank.Such surfactants may be obtained from the same chemical manufacturerslisted herein selling defoamers.

The fermentation tank is preferably sterilized between the growth ofdifferent batches of microbial growth, as yeast and other contaminantsmay be present. In one embodiment, the tank is sterilized using thespray bar mechanism comprising of the spray bar system for washing atthe waterline as described above and the sprinkler system, as explainedand illustrated in FIG. 4. The spray bar can be used to introducesterilization liquids, defoamer, or other liquids into the fermentationtank.

The tank may be cleaned with water, chlorine dioxide, or by exposure toUV light. Alternatively, the tank may be sterilized with an acidicsolution of pH 2 or less, such as sulfuric acid or phosphoric acid. Theacidic solution is then washed out of the tank, or in the case where thetank is sterilized with phosphoric acid, after sterilization, the acidmay be neutralized, retained in the tank, and used as a nutrient toprovide phosphorus for subsequent microbial growth. Alkaline, or verybasic, fluids can also be used for sterilization of the fermentationtank. While using either acidic or alkaline fluids, the tank may befilled to its operating level to obtain ideal and complete sterilizationprior to neutralizing the pH to grow the next batch of microbes.

The washing, cleaning, and sterilization steps can be accomplished asthree separate procedures, with three separate apparatus, such as spraybars, washing bars, and the like. These steps can also be accomplishedas three separate steps, or fewer combined steps using the sameapparatus. Additionally, variations or modifications of these procedurescan be used.

The fermentation system of the present invention is capable of growing asingle strain or multiple strains in a given batch. The system is alsocapable of growing multiple batches in a single day. In growing multiplestrains, care must be taken to accommodate for differences in growthrates between the various strains. Similarly, when growing multiplebatches, care must be taken to accommodate for differences in optimalgrowth parameters between batches.

Examples of both large scale and smaller scale fermentation systems areprovided below. A preferred embodiment of each a large scale and asmaller scale fermentation system are compared in the Table 3. Examples1-5 are generally directed to applications using the large scalefermenter, and examples 6-8 are generally directed to applications usingthe smaller scale fermenter.

TABLE 3 FEATURE LARGE SCALE SMALL SCALE Price more expensive lessexpensive Electric Supply 220 V or 240 V, 110 V, 220 V or 3 phase 240 V,single phase or 3-phase Operating Costs high electric low electric Waterwarm-up time 2-4 hours 8-16 hours Production time (Hrs/Batch) 8 hours16-24 hours Production time (Batches/day) 1-3 per day 1 per day Dosageduration 2 hrs 24 hrs to 7 days (Fill & draw cycle) Initial Inoculation(cfu/mL) 10⁶-10⁷ cfu/mL 10³-10⁴ cfu/mL pH control Yes Not RequiredAutomated Feed of Nutrients Optional Optional and Microbes RemoteMonitoring Optional No Pre-fermentation Optional Not RequiredPost-fermentation Optional Not Required Tank Size 500-1000 gallons 250gallons Plant size treated 10-40 MGD 0.1-10 MGD

Example 1 Large Scale System in General

One aspect of the current invention is the inoculation at theapplication point with about 10-100 times more of the target microbesthan conventional technology. With this in mind, the dosage at the pointof application is optimally at least about 10⁴ cfu/mL. Based on thisgoal, the working volume of the main fermentation tank is optimallyabout 1/500 to 1/50,000 of the flow to be treated, while the postfermentation tank is about 2-100 times larger in working volume, and thepre-fermentation tank is about 50-500 lesser in working volume. Themicrobial inoculum can be about 1/100 to 1/1000 of the first tank usedwhether that is the pre-fermentation tank or the main fermentation tank.For example, for a day supply of inoculum for a 40 MGD plant with apre-fermentation tank, the inoculum required to be stored would be 1.2gallons, or 5 gallons would last for about 3 months without replacement.

Table 4 shows the relationship between the quantity of flow to betreated and the inoculum required.

TABLE 4 Flow to be Pre- Main Post- treated Inoculum fermentationfermentation fermentation 40 MGD 0.04 gal/batch 4 gal 400 gal 4000 gal10 MGD 0.01 gal/batch 1 gal 100 gal 1000 gal  1 MGD 0.001 gal/batch  0.1gal    10 gal  100 gal

The specifications of this particular embodiment include the following.

Tank: fiberglass, high-density polyethylene (HDPE), steel.

Heating elements: 30-90 kW per 1000 gallons for preheat and internalheat depending on incoming water temperature.

Internal heating: 5-20 kW per 1000 gallons.

Preheating can be industrial in-line heaters, or domestic water heatingtanks.

Aeration: diffused air to achieve greater than 30 mg/L/hr transferefficiency.

With the feature of multiple daily cycles, the 400 gallon tank couldtreat the 10 MGD flow with 2.5 cycles per day, while a 1000 gallon tankcould treat the 40 MGD flow with 4 cycles per day.

The storage tank 14 used for automatic feeding of the mother culture, ororiginal culture, into the fermentation tank 12 is diagrammed in FIG. 1.This storage system/automated feeding tank 14 allows automaticinoculation of the fermentation tank 12. Initially, the inoculum isstored in the storage system/automated feeding tank 14. With propersterilization and cooling to about 4° C., a liquid mother culture may,optimally, be stored for a minimum of approximately 30 days. The mainfermentation tank 12 is then inoculated from the storagesystem/automated feeding tank 14. The main fermentation tank 12 may beinoculated with a portion of the stored mother culture for theinitiation of each batch of fermentation. In this case, the originalmother culture is the source of the inoculant. Alternatively, at the endof the fermentation process, a sufficient amount of the contents of themain fermentation tank 12 is removed back to the storage system 14 andsecond transfer tank (not shown), where the contents are held while theautomated feeding tank 14 is sterilized at the same time as the maintank 12. The main fermentation tank 12 is then emptied and sterilized.Then the contents of the second transfer tank are fed into thestorage/automated feeding system 14 and the transfer tank is thensterilized. The automated feeding tank 14, then feeds the contents backinto the main fermentation tank 12 to re-inoculate the next batch offermentation. In the case of reinoculation, the source of inoculant is aportion of the previous fermentation batch.

A liquid or a dry inoculum may be used to inoculate the fermentationtank 12. Liquid forms may be stored in a refrigerator to extend theshelf-life for a minimum of 10 days. Optimally, the alternative dryforms of preserved microbes have a shelf life of at least 6 months andare stored in sufficient quantity in the automated feed system thatmanual replacement of the dry forms of preserved microbes forinoculation of the fermentation system is only minimally required.Optimally, manual replacement is required not more often than once aboutevery 30 days. Where a pre-fermentation system is used, the dry formsmay be packaged in dissolvable, gel capsules for easier measurabilityand automated feeding.

The microbes used in the fermentation process may be exogenous,commercially available products or indigenous microbes isolated from thetreatment system.

The pre-fermentation tank 16 is illustrated in FIG. 1. The use of apre-fermentation tank 16 permits a reduction in the amount of inoculumrequired for the main fermentation tank 12 and the degree of storagerequired. For example, the main fermentation tank 12 is a 4000 gallontank, and sufficient inoculum is needed to achieve 10⁷ cfu/ml in the4000 gallon volume on a daily basis for 30 days. That would require, forexample, 40 gallons of a 10⁹ cfu/ml inoculum each day. Without the useof a pre-fermentation tank 16, this requires the storage of 1200 gallonsof inoculum for a 30 day supply. The use of a pre-fermentation tank 16,in which inoculum is grown from 10⁷ to 10⁹ cfu/ml, or 100 fold, allowsreduction of the stored inoculum 100 fold down to 12 gallons. In thiscase, 0.4 gallons of the 10⁹ cfu/ml culture are used to inoculate 40gallons each day. The 40 gallon volume is then grown from 10⁷ to 10⁹cfu/ml, or 100 fold, each day. The 40 gallon volume at 10⁹ cfu/ml isthen used to inoculate the 4000 gallon main fermentation tank 12 to astarting concentration of 10⁷ cfu/ml.

The use of a subsequent or post-fermentation tank 18, permits additionalflexibility and an increased dosage at the point of application, in amanner similar to that illustrated with the pre-fermentation tank 16.Inoculation from a post-fermentation system 18 or holding tank 20 ispreferable for single pass lagoon systems where there is no internalrecycle of the biomass, and hence inoculation of the incoming flow on acontinuous basis is critical to optimize the treatment.

A holding tank 20 is used when storage of the fermentation batch isdesired before the batch is delivered at the point of application.Although the post-fermentation tank 18 may be used as a holding tank 20,the holding tank 20 does not require heating, aeration, mixing, ornutrients supplied, as do the fermentation tanks.

An alternative mode of operation to batch fermentation is continuousfermentation. Generally, industrial manufacturing processes usingfermentation such as brewing, pharmaceuticals, and production of enzymesand microbes are batch fermentation processes. The reason industry hasadopted batch fermentation over continuous fermentation is thereliability of quality control and ability to easily deal with unwantedcontaminating microbes, which may ruin the end-product. In one aspect ofthis invention, continuous fermentation may be possible in certaincircumstances, where only one microbe is required and contamination canbe controlled for extensive lengths of time, for example in excess of10-30 days. Continuous fermentation would largely eliminate the need forpost-fermentation 18 or holding tanks 20 to feed the inoculumcontinuously. Alternatively, batches may be grown and continuously fedafter some predetermined time into the growth cycle, until the nextbatch was to be fed. For example, a batch could be grown for 6 hours,fed continuously for 18 hours gradually draining the tank, and theprocess started again the next day. Alternatively to gradually drainingthe tank, water, carbon sources, and nutrients could be introduced at aconstant rate, approximately equal to the rate at which inoculum isbeing fed to the application point with the whole tank being dumped atthe end of the fermentation cycle. This prevents washout or depletion ofthe microbes, so the microbes are not depleted in excess of theirability to grow to the desired concentration.

Example 2 System for Single Pass Lagoon System

In a single pass lagoon system, the influent is first directed to aprimary clarifier in which solids are allowed to settle. Then thewastewater is passed through an aerated lagoon, and then into a settlingpond, before discharge. In the single pass system, there is a continuousflow of wastewater, and therefore, continuous treatment is desired sothat each part of the waste steam is treated.

In a plant with a 14 MGD flow, a single pass lagoon system is inoculatedfrom a 1000 gallon tank containing a concentration of 1.4×10⁹ cfu/mlmicroorganisms. The initial concentration of microorganisms in theinoculated 4 million gallon aeration tank is 10⁵ cfu/ml. Typically 10⁵to 10⁷ cfu/ml microorganisms can be recovered from a single pass lagoon.The inoculum is therefore sufficient to obtain almost immediatetreatment. If the lagoon has a 3 day design residence time and theinoculum is added to the waste stream at the front end, then the effectis seen in the effluent in three days, or in the length of the residencetime. The effect is generally measured by determining the biologicaloxygen demand (BOD), chemical oxygen demand (COD), total organic carbon(TOC), or total carbon (TC) in the effluent.

NPDES permits for wastewater are generally in units of maximum allowedpounds per day (lbs./day) of BOD and a maximum monthly average. Thedaily maximum is usually two times the monthly average.

Without any treatment, the discharge from the plant releasesapproximately 30,000 lbs./day BOD. Optimally, a wastewater treatmentsystem reduces the BOD by approximately 90 percent. The permit for theplant sets limits at 6,000 lbs./day BOD as a monthly average and 12,000lbs./day BOD as a daily maximum. With the single pass lagoon systemcurrently in place, the plant is releasing wastewater with 7,000-15,000lbs./day BOD, and is not in compliance with the NPDES permit. Whentreatment is implemented with the fermentation system of the currentinvention, the effluent is down to 3,000 lbs./day BOD in 3 days.

The waste stream is dosed either at the front end of the lagoon, or atthe middle or back end of the lagoon, or at multiple sites.

In a single pass lagoon system, the dose of inoculum at the point ofapplication is optimally at least about 10⁴ cfu/ml. The concentration ofmicrobes at the point of application may be from about 10⁴ to 10⁸cfu/ml, alternatively it may be about 10⁵ to 10⁸ cfu/ml, or about 10⁶ to10⁸ cfu/ml, or about 10⁷ to 10⁸ cfu/ml, or about 10⁴ to 10⁷ cfu/ml, orabout 10⁴ to 10⁶ cfu/ml, or about 10⁴ to 10⁵ cfu/ml. The concentrationof microbes in the inoculant is optimally about 10⁹ to 10¹⁰ cfu/ml. Theconcentration of microbes in the inoculant may be about 10⁸ cfu/ml, orabout 10⁷ cfu/ml, or about 10⁶ cfu/ml. Depending on the dose of theinoculum, it may be possible to see a turn around in a single passlagoon system within about 5-7 days, or within less than about 5 days,or within less than about 4 days, or within less than about 3 days, orwithin less than about 2 days, or within about 1 day, or within theresidency time of the lagoon system.

Example 3 System for Activated Sludge System

In an activated sludge system, the influent is delivered to a primaryclarifier in which solids are allowed to settle. The wastewater thenpasses to an aerated basin, and then to a secondary clarifier wheresludge is recycled to pass through the aerated basin again. Due to therecycling in the activated sludge system, a holding tank is notnecessary, although it may be desired as a back up.

In a typical activated sludge system, it may take 14 to 30 days to seean effect of bioaugmentation due to the low rates of inoculation ofmicrobes. Using the multistage fermentation system of the presentinvention, an effect is seen in approximately 5 days. In a small plant(smaller wastewater flow) with a large fermentation tank, allowing alarger inoculum, an effect is seen in as little as 24 to 48 hours.

For example, a plant with an activated sludge system to treat itswastewater is treated using the fermentation system of the presentinvention. The plant has a 4 million gallon aeration tank which isinoculated from a 1000 gallon tank containing a concentration of 4×10⁹cfu/ml microorganisms. The initial concentration of microorganisms inthe inoculated 4 million gallon aeration tank is 10⁶ cfu/ml. Themicroorganisms contained in the inoculum have a generation time ofapproximately 30 minutes under ideal conditions, requiring 10 generationtimes, or about 5 hours, to grow 1000 fold. Typically, 10⁶ to 10⁷ cfu/mlcan be recovered from an activated sludge system containing a healthybiomass, and occasionally, a higher concentration can be recovered. Inthis case, the system can be turned around and brought into compliancewithin a day.

In an activated sludge system, the dose of inoculum at the point ofapplication is optimally at least about 10⁴ cfu/ml. The concentration ofmicrobes at the point of application may be from about 10⁴ to 10⁸cfu/ml, alternatively it may be about 10⁵ to 10⁸ cfu/ml, or about 10⁶ to10⁸ cfu/ml, or about 10⁷ to 10⁸ cfu/ml, or about 10⁴ to 10⁷ cfu/ml, orabout 10⁴ to 10⁶ cfu/ml, or about 10⁴ to 10⁵ cfu/ml. The concentrationof microbes in the inoculant is optimally about 10⁹ to 10¹⁰ cfu/ml. Theconcentration of microbes in the inoculant may be about 10⁸ cfu/ml, orabout 10⁷ cfu/ml, or about 10⁶ cfu/ml. Depending on the dose of theinoculum, it may be possible to see a turn around in an activated sludgesystem within about 5-7 days, or within less than about 5 days, orwithin less than about 4 days, or within less than about 3 days, orwithin less than about 2 days, or within about 1 day, or in less than 1day.

Example 4 Automated Fermentation System

One embodiment of an automated system 50 is shown in FIG. 5. In thissystem there is a storage tank 50 for holding and maintaining theinoculum (this tank may also be referred to as the inoculum tank), afermentation tank 54, and a control unit 56.

The inoculum tank 52 is used to hold and maintain the inoculum, whichcontains the microbe that is being used to treat the waste water system.The tank 52 is intended to hold and maintain inoculum for at least aweek, at least a month, and perhaps longer than a month. Optimally, thetank 52 is covered and temperature controlled. The tank may be made fromany material known to those skilled in the art that is compatible forsuch uses. For example, the inoculum tank 52 may be made fromfiberglass, high-density polyethylene or steel. Optimally, the tank hasa probe or probes 58 that measure temperature, oxygen level, pH,turbidity, conductivity, ammonia and other conditions necessary tomaintain for optimal growth or that show the amount and viability of themicrobes that make up the inoculum. The signals from the probe or probes58 are then communicated to the control unit 56, which monitors andcontrols the system. A pump 60 is associated with the inoculum tank 52.This pump 60 is used to transfer the inoculum to the fermentation tank54. The pump 60 has relays, sensors, or monitors that are incommunication with the control unit 56. The control unit 56 monitors andoperates, i.e., turns on and off the pump. The pump 60 is any suitablemetering pump. In this way, the time of pump operation can be equated toa specific volume of inoculant transferred to the fermentation tank 54.Although the embodiment in FIG. 5 shows a single inoculum tank 52 itshould be understood that multiple inoculum tanks may be used to havethe ability of having different microbes as the inoculant, or in thealternative, a mixture of microbes may be used in a single inoculum tank52.

The fermentation tank 54 is generally larger than the inoculum tank 52.The fermentation tank 54, as with the inoculum tank, optimally iscovered. The fermentation tank 54 preferably has a spray bar 62, a feedline 64 to the spray bar, a water feed line 66, a probe or probes 68, aheating element 70, an aeration element 72, and a nutrient feed line 74.

One embodiment of the spray bar 62 is illustrated in greater detail inFIG. 4. The spray bar 62 is used to deliver various liquids into thefermentation tank 54. Such liquids include, but are not limited to,liquids used for washing or sterilizing the fermentation tank, liquidsfor neutralizing or controlling the pH of the contents of thefermentation tank, or defoamer. The liquid is provided to the spray bar62 through the feed line 64, and the flow in to the feed line 64 can becontrolled by a valve 80. The water line 66 is used to deliver waterinto the fermentation tank 54. The flow into the water line 66 can becontrolled by a valve 78.

A probe or probes 68 can be positioned external to the fermentation tankand flow may be delivered by a pump recycling the contents of thefermentation tank 54 past the probe or probes 68, such that the probes68 are able to detect various characteristics of the contents of thefermentation tank 54. Alternatively, a probe or probes 68 may be locatedwithin the tank 54 for detecting various characteristics of thecontents. The probes 68 may detect temperature, pH, oxygen levels,conductivity, turbidity, nutrient levels, or other variables useful inmonitoring and controlling the growth of the microbes. Optimally, theprobes 58, 68 are in communication with the control unit 56 so that thecontrol unit 56 can be used to send an appropriate signal to cause achange in the growth conditions in the fermentation tank 54. Forexample, the probes 68 may be in communication with the control unit 56,which in turn may control the valves and other devices which regulatethe temperature, pH, nutrients, and other growth parameters.

The heating element 70 is used to control the temperature of thefermentation tank 54. Depending on the conditions, and whether heatingor cooling is required, this element 70 may be a cooling rather thanheating element. Additionally, instead of an element 70, a jacketed tankmay be used, or other types of temperature control apparatus may beused.

Air is supplied through a gas transfer system or aeration element 72which may deliver oxygen into the fermentation tank 54, as in the caseof growing aerobic microbes. The aeration may provide sufficient mixingof the contents of the fermentation tank 54. Alternatively, mixing maybe provided by a separate mixing device 86. Optimally, the oxygen levelsin the fermentation tank 54 are controlled. In the case of growinganaerobic microbes, the gas transfer system 72, instead of deliveringoxygen to the fermentation tank 54, is used to pull gas out of thefermentation tank headspace via connection to a pump (not shown).

A nutrient feed line 74 is used to deliver nutrients, includingmicro-nutrients, macro-nutrients, and carbon sources, into thefermentation tank 54. The flow of nutrients into the nutrient feed line74 may be controlled by a valve 76.

The flow of the inoculum out from the fermentation tank 54 is controlledby a pump 82. The inoculum may flow from the fermentation tank 54 toanother tank or tanks (not shown), to the point of application, or tosome other location. The flow of the inoculum from the fermentation tank54 back into the inoculation tank 52, is controlled by pump 84. At theend of a fermentation batch, an amount of the batch may be redeliveredback into the inoculation tank 52 to be used to inoculate a subsequentmicrobial growth batch.

Example 5 Automated Multi-Tank Fermentation System

One embodiment of a multistage tank system 90 of the present inventionis illustrated in FIG. 6. In this system there is a storage tank 92 forholding and maintaining the inoculum, this tank may also be referred toas the inoculum tank 92, a fermentation tank 94, a control unit 96,which monitors and controls the system, a pre-fermentation tank 100, apost- or subsequent fermentation tank 102, and a holding tank 104. Thisembodiment, employing a series of fermentation tanks permits a reductionin the amount of inoculum required to be stored in order to operate thefermentation system, because the inoculum is allowed to grow through agreater number of generations before delivery at the point ofapplication.

As in Example 4, a pump 98 is associated with the inoculum tank 92. Thispump 98 is used to transfer the inoculum to the fermentation tank 94, orto the pre-fermentation tank 100, or to a combination of the two. Avalve 114 controlling the flow from the inoculum tank 92 to thepre-fermentation tank 100 and a valve 116 controlling the flow from theinoculum tank 92 to the main fermentation tank 94, allows control of theflow of inoculum. When the valve 114 controlling the flow from theinoculum tank 92 to the pre-fermentation tank 100 is open, the inoculumflows to the pre-fermentation tank 100 when the pump 98 associated withthe inoculum tank 92 is on. The inoculum is allowed to grow in thepre-fermentation tank 100 for a number of generations. There is a pump106 associated with the pre-fermentation tank 100 which controls theflow of inoculum from the pre-fermentation 100 tank to the mainfermentation tank 94.

From the main fermentation tank 94, the inoculum may flow into apost-fermentation tank 102, a holding tank 104, a point of application,or back into the inoculum tank 92. There is a pump 108 associated withthe main fermentation tank 94 for controlling flow out of the mainfermentation tank 94.

For flow from the fermentation tank 94 into the post-fermentation tank102, the main fermentation tank-associated pump 108 is on and the valves122, 124 leading from the main fermentation tank 94 to thepost-fermentation tank 102 are open.

For flow from the fermentation tank 94 into the holding tank 104, themain fermentation tank-associated pump 108 is on and the valves 122, 130leading from the main fermentation tank 94 to the holding tank 104 isopen. If flow is desired only into the holding tank 104, and not thepost-fermentation tank 102, the valve 124 controlling flow into thepost-fermentation tank 102 is closed.

For flow from the fermentation tank 94 to the point of application, themain fermentation tank-associated pump 108 is on and the valves 120, 136leading from the main fermentation tank 94 to the point of applicationare open.

For flow from the fermentation tank 94 to the inoculum tank 92, the mainfermentation tank-associated pump 108 is on and the valves 118, 120leading from the main fermentation tank 94 to the inoculum tank 92 areopen. If flow is desired only from the fermentation tank 94 to theinoculum tank 92, the valves 122, 136 controlling flow from thefermentation tank 94 to the post-fermentation tank 102, the holding tank104, and the point of application are closed.

The inoculum can be transferred from the post-fermentation tank 102 tothe holding tank 104 or to the point of application. There is a pump 110associated with the post-fermentation tank 102.

For flow from the post-fermentation tank 102 to the holding tank 104,the post-fermentation tank-associated pump 110 is on, and the valve 128which controls the flow from the post-fermentation tank 102 to theholding tank 104 is open. If flow is desired only from thepost-fermentation tank 102 to the holding tank 104, the valve 126 whichcontrols the flow from the post-fermentation tank 102 to the point ofapplication is closed.

For flow from the post-fermentation tank 102 to the point ofapplication, the post-fermentation tank-associated pump 110 is on, andthe valve 126 which controls the flow from the post-fermentation tank102 to the point of application is open. If flow is desired only fromthe post-fermentation tank 102 to the point of application, the valve128 which controls the flow from the post-fermentation tank 102 to theholding tank 104 is closed.

The inoculum can be transferred from the holding tank 104 to the pointof application or to the post-fermentation tank 102. There is a pump 112associated with the holding tank 104 for controlling flow out of theholding tank 104.

For flow from the holding tank 104 into the post-fermentation tank 102,the holding tank-associated pump 112 is on and the valve 134 leadingfrom the holding tank 104 to the post-fermentation tank 102 is open. Ifflow is desired only into the post-fermentation tank 102, and not thepoint of application, the valve 132 controlling flow from the holdingtank 104 to the point of application is closed.

For flow from the holding tank 104 to the point of application, theholding tank-associated pump 112 is on and the valve 132 leading fromthe holding tank 104 to the point of application is open. If flow isdesired only to the point of application, and not to thepost-fermentation tank 102, the valve 134 controlling flow from theholding tank 104 to the post-fermentation tank 102 is closed.

A smaller scale fermenter is used primarily for non-emergency upsetconditions and general maintenance of biomass health for the applicationpreviously described for the large fermentation system including BODremoval and filamentous bulking control. Individually, such a system canbe used, for example, to treat smaller treatment systems, grease traps,drain-lines, lift stations, and septic tanks, or several systems can bedeployed to treat a large lagoon system with a large flow. Thefermentation process can also be used to grow batches of microbes whichare harvested and dispensed into smaller containers, such as I galloncontainers, for use as a drain line maintenance product for grease trapsor bioremediation or starter culture for septic tanks. Larger 5-50gallon batches or the whole 250 gallon tote can be deployed by servicecompanies operating wastewater treatment systems (municipal orindustrial), which have lift stations in which grease build-up is aproblem. Service companies of Hazmat teams can also use the smallersystem to bioremediate small spills of organics, contaminated sites orunderground storage tanks containing contaminated water.

Generally, a fermenter of this type includes a tank, aeration, and acontroller. In one embodiment of a smaller scale fermenter, the tank ispreferably a 250 gallon tote that is skid mounted, moveable, andshippable. The aeration pump is preferably exterior to the tank, and anaspirator such as an Eco aspirator from Aquatic Eco-Systems Inc. mayoptionally be used. The controller in this particular embodiment is amodular controller comprising a timer, relays for switching devices onand off, a manual on-off switch, a temperature sensor and floatconnected to heating elements to heat the liquid, which connects to aregular domestic electricity supply such as 110V, 230V or 460V in theUSA, or 240V or 480V in Europe. Internal electronic circuits can beoperated off 12V using a transformer or electronic device to step downthe voltage. A discrete or continuous level sensor device with an liquidcrystal display (LED) can also be added to the modular controller tocontrol the process primarily by monitoring the level of liquid ratherthan by time. The modular controller controls the fill and drain cycleand mixing and aeration by opening and closing solenoid valves. Heatingelements are switched on and off according to temperature, and forsafety purposes at low level using a float or other level-measuringdevice such as a pressure transducer. Temperature can be controlledusing a fixed or variable thermostat. A pump is used to recirculate thecontents, optionally through an aspirator (preferably, with no movingparts), for efficient aeration and trouble-free operation. The pump alsoacts to discharge part or all of the contents of the tank when the timeropens the discharge circuit.

The smaller scale fermenter can be used as a manual system with anoptional upgrade to automate various features for the filling cycle,draining, and feeding of nutrients and microbes. The smaller scalefermenter can also be used for bioremediation of soil at contaminatedsites or for spill response of Hazmat Teams or bioremediation of organiccontaminants in underground storage tanks. For small bioremediation jobsof less than 100 cubic yards or the other applications described above,a smaller system consists of a shippable, watertight, 5-gallon plasticcontainer as the fermentation tank, an initial microbial inoculum in agelatin capsule, a pack of nutrient, an aquarium heater, air pump anddiffuser for aeration. The fermentation tank is filled with 5-gallons ofcity water and the nutrient pack and microbial inoculum are added. Theaquarium heater is inserted inside the container along with air stonesto diffuse air through the liquid to provide oxygen and mixing. Thecontents is preferably fermented for 8-24 hours before use.

Another example of an application of the small automated system is fortreating grease traps or grease build-up in drain lines and as a generaltreatment method for drain line maintenance for removing and reducingaccumulation of organic matter. This system comprises a controller and achamber for growing microbes with a mixing and/or aeration device, suchas an air stone and air pump for aeration and mixing with an automateddispensing system of microbes and nutrient in a single gel capsule or inseparate capsules. The controller controls the addition of city waterand the dispensing of the capsules into the fermentation tank. Thefermentation process then proceeds at room temperature over a 18-24 hourperiod before the contents are flushed into a drain line, for example,in a drain line of a restaurant after the restaurant is closed or aftersanitization of the kitchens is complete.

The size of these smaller fermentation tanks, chambers or vessels arepreferably about 0.25 gallons to about 250 gallons, with typical sizesof 5 for small bioremediation applications, 55 and 250 US gallons forwastewater treatment systems, lift stations and large bioremediationsites, and preferably about 1 liter for drain line maintenance atrestaurants.

These small 110V systems can be deployed more economically than thelarger automated systems. For example, four individual 250-gallonsystems are deployed to treat flows of 10-40 MGD. This is particularlyuseful where the systems are single pass lagoons covering a number ofacres. In such a case, several dosing points allow treatment to proceedsimultaneously across a lagoon which could hold 10-20 days of waterwhich does not meet the environmental requirements for discharge. Forexample, in a lagoon or holding pond, three 250-gallon fermentationsystems could be set-up at equidistant intervals from the back of thebasin with the last system treating a volume or area equivalent to onedays discharge of water. A fourth 250-gallon tank would be placed at thefront of the system to treat the new incoming wastewater.

Nutrients and microbes can be packaged in gelatin capsules or in watersoluble bags. Preferably the nutrients are packaged in water-solublebags, and the microbes are packaged in gelatin capsules. Packages ofnutrients and of microbes can be purchased from Advanced BiologicalServices Inc, Duluth, Ga. (www.waste-water.com). Sterilizing andcleaning agents such as sodium hypochlorite solution (i.e. bleach) orcommercially available cleaners in solid form can also be packagedwater-soluble bags or capsules. Liquid cleaners can be packaged in smalldispensing bottles. These cleaners and sterilizing agents are used bythe operator to sterilize or minimize contamination build-up over timein the fermentation process. For example, the tank can be filled withwater and a 0.5 to 1 lb. bag can be applied to 250 gallons of water andleft for 1 hour. The contents of the tank can then be emptied and thetank flushed with clean water before filling with water used to grow themicrobes.

Example 6 Daily Batch of 250 Gallons Treating 2-10-MGD Flow-Manual

As illustrated in FIG. 7, the fermentation tank 152 is filled manuallywith 250 gallons of water by opening valve 154. The water can be citywater, water from the factory, non-contact cooling water, primaryeffluent with low solids of less than 50 mg/L total suspended solids(TSS), or final effluent with less than 50 mg/L (TSS). There is a mainon-off control box 156 for power to the heaters and pump. As the waterreaches the 250-gallon mark on the tank 152 the operator closes watervalve 154 to stop water entering. As the tank 152 is filling, the heaterelements 158 come on at about 10-15% of full (25-30 gallons). Theoperator then adds 2-10 lbs of nutrient 160, preferably in water-solublebags (individually weighing 0.5, 1, or 2.5 lbs each. The nutrient bagscan contain dissolvable gelatin capsules of 0.5, 1, 2 or 3 oz sizecontaining the microbial inoculum 162 as well or be packaged separately.The pump 164 recirculates the water, nutrient and microbe mix,optionally through an aspirator 166 which draws air in through a tube168 and which can be located inside or outside the tank 152. The batchis allowed to ferment for at least 18-24 hours before dumping the batchto treatment system. The operator returns the following day and opensthe discharge valve 170 and closes the recirculation valve 172.Alternatively this can be a 3-way valve. This causes the recirculatingpump 164 to discharge the contents of the tank 152 or batch. As the tank152 empties, the operator may open water valve 154 to flush the tank 152and clean it out. On completion of emptying the tank 152 and washing itout the operator closes the discharge valve 170 and opens therecirculation valve 172.

Example 7 2-7 Day Cycle—1 Batch of 250 Gallons Treating 0.01-2MGD-Manual

As illustrated in FIG. 7, the fermentation tank 152 is filled manuallywith 250 gallons of water by opening valve 154. The water can be citywater, water from the factory, non-contact cooling water, primaryeffluent with low solids of less than 50 mg/L total suspended solids(TSS), or final effluent with less than 50 mg/L (TSS). There is a mainon-off control box 156 for power to the heaters and pump. As the waterreaches the 250-gallon mark on the tank 152 the operator closes watervalve 154 to stop water entering. As the tank 152 is filling, the heaterelements 158 come on at about 10-15% of full (25-30 gallons). Theoperator then adds 2-10 lbs of nutrient 160, preferably in water-solublebags (individually weighing 0.5, 1, or 2.5 lbs each. The nutrient bagscan contain dissolvable gelatin capsules of 0.5, 1, 2 or 3 oz sizecontaining the microbial inoculum 162 as well or be packaged separately.The pump 164 recirculates the water, nutrient and microbe mix,optionally through an aspirator 166 which draws air in through a tube168 and which can be located inside or outside the tank 152. The batchis allowed to ferment for at least 18-24 hours before application. Theoperator returns the following day and opens the discharge valve 170 andcloses the recirculation valve 172. Alternatively this can be a 3-wayvalve. This causes the recirculating pump 164 to discharge the contentsof the tank 152. The operator can dispense a certain portion of the tank152 contents according to the schedule of treatment which could be onebatch every 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9days, 10 days, 11 days, 12 days, 13 days, 14 days or longer. However,the maximum growth will occur in about 24 hours without further additionof nutrients and it is therefore recommended that optimally batches befed within 7 days or more nutrient added. When the tank 152 is empty theoperator may open valve 154 to flush the tank 152 and clean it out. Oncompletion of emptying the tank 152 and washing it out the operatorcloses the discharge valve 170 and opens the recirculation valve 172.

Example 8 1 Batch of 250 Gallons Treating 0.05-10 MGD Flow-Fill andDrain Cycle Automated

As illustrated in FIG. 8, the fermentation tank 182 is filledautomatically with 250 gallons of water at the beginning of the cycle bythe opening of a solenoid valve 184 controlled by an electronic board ortimer such as a garden sprinkler system 186 through a relay. As thewater reaches the 250-gallon mark on the tank a high level float 188closes valve 184 to stop water entering. As the tank 182 is filling theheater elements 190 come on at about 10-15% of full (25-30 gallons). Theoperator then adds 2-10 lbs. of nutrient 192 un water-soluble bags (5)individually weighing 0.5, 1 or 2.5 lbs. each. The nutrient bags 192 cancontain dissolvable gelatin capsules of 0.5, 1, 2 or 3 oz. sizecontaining the microbial inoculum 194 as well or be packaged separately.The pump 196 recirculates the water, nutrient and microbe mix,optionally through an aspirator 198 which draws air in through a tube200 and which can be located inside or outside the tank 182. The batchis allowed to ferment for at least 18-24 hours before dispensing part orthe entire batch to a treatment system. The controller 186 opens thedischarge solenoid valve 202 and closes the recirculation solenoid valve204, which causes the pump 196 to discharge the contents of the tank 182or batch on a timed or level controlled basis. On completion of thecycle and emptying the tank 182, the operator returns to wash it out.

Example 9 Weekly Cycle—1 Batch of 250 Gallons Treating 0.5-1 MGD-Manual

As illustrated in FIG. 7, the fermentation tank 152 is filled manuallywith 250 gallons of water by opening valve 154. The water can be citywater, water from the factory, non-contact cooling water, primaryeffluent with low solids of less than 50 mg/L total suspended solids(TSS), or final effluent with less than 50 mg/L (TSS). There is a mainon-off control box 156 for power to the heaters and pump. As the waterreaches the 250-gallon mark on the tank 152 the operator closes watervalve 154 to stop water entering. As the tank 152 is filling, the heaterelements 158 come on at about 10-15% of full (25-30 gallons). Theoperator then adds 4 bags of nutrient 160, 4 water soluble capsulescontaining the microbial inoculum 162, and 1 quart of defoamer. Eachnutrient bag contains 2.5 lb of nutrient in a water-soluble bag. Thecapsules containing the microbial inoculum can be add separately orpackaged with the nutrient bags. The pump 164 recirculates the water,nutrient and microbe mix, optionally through an aspirator 166 whichdraws air in through a tube 168 and which can be located inside oroutside the tank 152. The batch is allowed to ferment for at least 18-24hours before application. The operator returns the following day, closesthe recirculation valve 172 and opens the discharge valve 170.Alternatively this can be a 3-way valve. This causes the recirculatingpump 164 to discharge the contents of the tank 152. The operatordispenses a portion of the tank 152 contents according the schedule oftreatment of one batch every 7 days. It is recommended that batches befed within 7 days or more nutrient added. On completion of dispensing aportion of the tank 152, the operator opens the recirculation valve 172and closes the discharge valve 170. When the tank 152 is empty theoperator may open valve 154 to flush the tank 152 and clean it out. Oncompletion of emptying the tank 152 and washing it out the operatorcloses the discharge valve 170 and opens the recirculation valve 172.

This procedure can be adapted for treating larger flows of water. Ingeneral, treatment of a larger flow or of more severe upsets oroperational problems requires the discharge of a larger portion of thetank or an increase in the dosing rate. For example, the operator candispense a certain portion of the tank 152 contents according to theschedule of treatment which could be one batch every day, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days,12 days, 13 days, 14 days or longer. However, the maximum growth willoccur in about 24 hours without further addition of nutrients and it istherefore recommended that optimally batches be fed within 7 days ormore nutrient added. The fermentation tank 152 may optionally beequipped with an automated discharge system. For example a positivedisplacement pump controlled by a timer can provide for automaticopening and closing of the discharge valve 170 and the recirculationvalve 172 (or a 3-way valve, if present). Once the initial fermentationis complete, the automatic discharge system may discharge a knownquantity of the tank 152 intermittently or may discharge a constant flowon a continuous basis. Table 5 shows examples of the relationshipbetween the quantity of flow to be treated, the time per batch and thedosing rate.

TABLE 5 Flow to be treated Time per Batch Dosing Rate (gal/day) 10-20MGD 1 day  250 5-10 MGD 2 days 125 2-5 MGD 3 days 80 1-2 MGD 5 days 500.5-1 MGD 7 days 35 <0.5 MGD 10 days  25

Example 10 Cleaning of Fermentation Tank

The fermentation tank (152 or 182, FIGS. 7 and 8) can be cleanedperiodically, preferably once a month. To carry out a cleaning cycle,the tank is completely discharged, and the top of the tank is opened. Ifa low level float is present, the float can be disabled or the basecontents of the tank can be flushed out with water to dilute anyremaining fermentation product. If the tank can be operated in anautomatic or manual mode, it should be switched to the manual mode. Thedischarge sequence is started by engaging the main on/off switch. Thetank is completely filled with water. During the filling of the tank,the on/off switch is engaged again to close the discharge valve and openthe recirculation valve, and 5 gallons of bleach are added. Once thetank is filled, the bleach solution is recirculated for 2 hours. Afterthis soak time, the on/off switch is engaged again to start thedischarge sequence. The tank is rinsed and is then ready for use.

Example 11 Treatment of Grease in Wet Wells

Small scale manual fermentation systems were used to inoculate sewersystem wet wells with fat, oil and grease (FOG) degrading microbes. Fourcategories of wet wells were defined based on flow (low or high), freewater on the surface, thickness of the FOG cap and the degree and/orrate of accumulation of FOG on the surface of the water. Well A wasdefined as a low flow wet well with a moderate grease accumulation of6-8 inches on the water surface. This well had only 20% or less freewater, and the floats in the well were grounded. Well B was defined as alow flow wet well with a severe grease accumulation of 12-18 inches onthe water surface. This well had little, if any, free water, and thefloats in the well were grounded. Well C was defined as a low flow wetwell with a moderate to severe grease accumulation of 8-10 inches and20% or less free water. This well also was showing signs of a rapidincrease in grease accumulation. Well D was defined as a high flowpumping station with a severe grease accumulation of 18-24 inches. Thiswell had a water flow rate of 5 MGD.

The microbes used for each wet well were chosen to provideemulsification and degradation of the FOG into carbon dioxide and water.A generic blend of microbes for degrading FOG was used for Wet Well Aand a specific culture from Novozymes called Bi-Chem LC 2009 GT for WetWell B. Wet Well A was dosed at a frequency of 250 gallons per week forthe first two weeks and then 250 gallons over 4 weeks thereafter. WetWell B was defined as a more severe application and was dosed at 500gallons per week. A spray distribution system was employed toeffectively distribute the discharged inoculum onto the FOG.

Within 6 weeks of the beginning of treatment, Wet Well A had shown anincrease in free water levels of approximately 70%, thereby nothindering floats controlling the pumps. The remaining FOG became freefloating and no longer forming a hard mat across the surface of the wetwell. Wet Well B became more soupy but never showed signs of waterbreaking. In this case it was decided that the application forcommercial purposes would require the wet well to be pumped out prior toongoing treatment so that the FOG degradation program could keep up withthe rate of accumulation rather than trying to overcome historicbuild-up. The degradation of the FOG provides for a soupy mixture oforganics, microbes and water. This mixture can then be pumped throughthe existing water lines, and in some cases the organics will dissolveor disperse in the water altogether. The residual microbes can continueto degrade grease throughout the sewer lines as the mixture is pumpedaway from the wet well. The microbes will also tend to cling to thewalls and other structures causing retention in the wet well of suchmicrobes.

There are many applications and numerous embodiments of the presentinvention. In addition to the embodiments specifically discussed above,some of the additional embodiments or characteristics of thoseembodiments are described below.

In one embodiment of the invention there is a fermentation systemcomprising a closed fermentation tank (with an air vent), with a spraybar and sprinkler system for introducing a fluid for sterilization ofthe tank, in which the contents are aerated, heated, mixed andmonitored. The process can be a multi-staged fermentation process usingtanks of increasing size.

The fermentation tank is optionally skid mounted and moveable by aforklift truck. The tank can also be weatherproof to the point where itcan be located outside or onsite at an industrial facility without theneed for further enclosure to prevent loss of functionality.

Aeration for the tank can be provided by a dedicated blower, or by usingplant air at a manufacturing site. Pure oxygen can be introduced toprovide supplemental oxygen through the aeration device into thefermentation tank. Air can be introduced through a fine bubble diffusersystem to satisfy oxygen uptake rates of greater than 10 mg/L/hr, ofgreater than 20 mg/L/hr, or of greater than 30 mg/L/hr. Preferably, theaeration creates sufficient mixing to maintain the microbes insuspension. In one embodiment, the aeration device is tubular and madeof vyon. The electrical energy costs for operation can by minimized byreducing the amount of aeration required for treatment.

The desired temperature of the tank depends on the type of microbe ormicrobes being grown. In one embodiment, the water for making up thecontents of the tank is preheated to raise the temperature a minimum ofabout 10° C. in about one hour or less, either using domesticallyavailable water heaters or commercially available industrial in-lineheaters. Alternately, the water can be preheated to raise thetemperature about 25° C. in about one hour or less.

The contents of the tank can be either heated or cooled to bring thetemperature into an optimum range for fermenting and growing themicrobes, and the tank can be subsequently maintained at such atemperature. For example, the contents of the tank can be cooled tobring the temperature into the range of about 15° C. to about 18° C. forpsychrophilic microbes. The contents of the tank can be heated to bringthe temperature into the range of about 30° C. to about 37° C. formesophilic microbes, or into the range of about 45° C. to about 80° C.for thermophilic microbes.

The fermentation process can be monitored either on site or remotely. Inone embodiment, the contents of the fermentation tank are monitoredduring the fermentation process locally with an option to access andcontrol the fermentation process from a remote location. The remotemonitoring can occur where some control systems may be in the samebuilding or the system may include an option to access and control thefermentation process from a remote location more than 1 mile away. Thegrowth of microbes can be monitored during the fermentation by changesin turbidity and/or conductivity, and additional nutrients, carbonsources, or selective carbon sources can be automatically added or thetemperature can be adjusted. The pH of the tank contents can also bemonitored and automatically controlled to optimize the fermentationprocess.

The fermentation tank is preferably sterilized between the growth ofdifferent batches. The sterilization agent can be either highly acidic(phosphoric acid) or highly alkaline (caustic). The sterilization agentcan also be used as a nutrient in the fermentation process, for example,phosphoric acid can be used as a sterilization agent and also provide anutrient used in the fermentation process. The sterilization agent canalso be used to control the pH of the contents of the fermentation tank,for example, phosphoric acid can be used as a sterilization agent andalso used to control the pH of the contents of the fermentation tank.

In one embodiment, at the end of the fermentation process, a sufficientamount of the contents of the fermentation tank is removed to a holdingtank or tanks which serve to hold the microbes while the fermentationtank is emptied and sterilized. After sterilization, the contents areautomatically dumped into the main fermentation tank in order tore-inoculate the contents and initiate the next batch of microbes. There-inoculation of microbes for the next batch can continue withoutmanual intervention or a new mother culture for a period of at leastabout 5 days continuously, or for a period of about 30 dayscontinuously.

The microbes used to initiate the batch can be grown in a smallerpre-fermentation tank about 50-100 times smaller than the mainfermentation tank. The microbes grown in the pre-fermentation tank canbe grown daily. The inoculum for the pre-fermentation tank can beautomatically fed in the form of a fluid from a reservoir or storagevessel retaining the mother culture. Additionally, storage of the fluidmother culture can be in a refrigerated compartment to extend itsefficacy for at least about 30 days.

As an alternative to a liquid mother culture, a dried culture can alsobe used for inoculation, The inoculum for the pre-fermentation tank canbe automatically fed in the form of freeze dried microbes, with orwithout a carrier, contained in soluble capsules, such as gelatin, or asanother form of preserved, dry material such as flakes. Alternative dryfauns of preserved microbes can have a shelf life of at least about 6months and can be stored in sufficient quantity in the automated feedsystem that manual replacement of the dry forms of preserved microbesfor inoculation of the pre-fermentation system is required not moreoften than about once every 30 days. In another embodiment, dry forms ofpreserved microbes can have a shelf life of at least about 12 months andcan be stored in sufficient quantity in the automated feed system thatmanual replacement of the dry forms of preserved microbes forinoculation of the pre-fermentation system is required about once every12 months.

The fermentation system can also be adapted to a treatment system, inpart, by the immobilization of the microbes on a fixed-film media, suchas looped cord media, plastic packing, or other inert, high surfacemedia on which microbes attach.

The microbes used in the fermentation process can be exogenous,commercially available products or they can be indigenous microbesisolated from the treatment system. The fermentation process can beadapted to grow different strains, types, or species of microbes duringdifferent cycles of the process. The fermentation process can be used togrow aerobic, facultative, or anaerobic microbes. The fermentationprocess can be used to grow lithotrophic microbes, or nitrifiers, suchas Nitrosomonas and Nitrobacter, to establish and maintain nitrificationin a biological wastewater treatment system.

In one embodiment, the contents of the fermentation system are used toinoculate a post-fermentation tank about 5-100 times larger than themain fermentation tank prior to being fed into the biological wastewatertreatment system. The contents of the fermentation system can also bepumped into a holding tank or tanks to be transferred to the biologicaltreatment system over the cycle period of the fermentation tank.

The fermentation system can be used for multiple cycles, or grow morethan one batch per day, to ferment several different microbes, or toincrease dosage rates or dose different microbes, and optionally, aholding tank can be used for each microbe. The inoculation of thefermented cultures into the biological system can occur on a dailybasis, it can occurs more than once a day, or it can occur less oftenthan daily. The inoculation from a post-fermentation system or holdingtank can be used for single pass lagoon systems where there is nointernal recycle of the biomass, and it is desirable to continuouslyinoculate the incoming flow of waste water.

The fermentation process can be used to treat aqueous liquids, includingwastewater, ground water, and aqueous liquids in systems such as wetwells, drain lines, septic tanks, and underground storage tanks. Forexample, the inoculum can be injected into a ground water aquifer. Thefermentation process can also be used to treat contaminated soil. Forexample, the inoculum can be applied on the soil surface, or theinoculum can be mixed with the soil surface. An aqueous liquid, bydefinition, is a liquid containing water, and includes pure water,contaminated water, and water containing other dissolved or dispersedsolids or liquids (i.e. emulsions, solutions, dispersions). Water istypically the major component of an aqueous liquid, although the termincludes liquids having only a minor component of water.

The fermentation process can be used to treat an accumulation of fat,oil and/or grease (FOG) in sewer system wet wells. The inoculum from thefermentation process can be applied to a wet well on a temporary basisto reduce or eliminate the FOG. The inoculum from the fermentationprocess can also be applied to a wet well on a continuous basis tomaintain the FOG accumulation below a desired level.

In the treatment of relatively small and isolated systems, such asmultiple wet wells or multiple areas of contaminated soil, it may bedesirable to use portions of a single inoculum from the fermentationprocess to treat more than one site. For example, the inoculum from thefermentation process may be transferred to a mobile container, such as atank on a truck equipped with the appropriate pumps and liquiddistribution equipment. The inoculum can then be transported to eachtreatment site, and a portion of the inoculum can be administered to thesite. In another example, a small fermentation system may be mounted ona truck or trailer. The fermentation system can then be transported toeach treatment site, and a portion of the inoculum can be administeredto the site from the fermentation system.

The fermentation process can be used to treat wastewater that has notyet been transferred into a biological wastewater treatment system. Forexample, the inoculum from the fermentation process can be applied to awastewater in a primary clarifier.

The fermentation process can be applied to start up a biologicalwastewater treatment system rapidly so that the manufacturing processcan continue without interruption due to restrictions caused by NPDEScompliance, or by compliance with other environmental regulations of thebiological wastewater treatment plant.

The production of hydrogen sulfide by sulfate reducing bacteria (SRB's)under anaerobic conditions causes major industrial problems, from odorsin wastewater treatment plants, to odors in sewer lines, corrosion ofsewer lines and metallic objects or structures exposed to the hydrogensulfide. This fermentation process can be applied using microbes tostabilize the hydrogen sulfide to elemental sulfur or to sulfuric acid,generally a less desirous end-product because of issues associated withcorrosion due to low pH. Microbes such as Thiosphera pantotropha (seealso Table 1) or purple sulfur bacteria can be grown in a fermentationsystem and applied to the waste stream or anaerobic plant where thehydrogen sulfide is formed or where is evolves into the gaseous state.One such application would be in an ammonium sulfite/sulfate mill, suchas Inland in New Johnsonville, Tenn. where sulfite/sulfate becomesconverted to hydrogen sulfide upon entering the anaerobic pond. Thesulfide levels in the anaerobe pond subsequently reach 800-1000 mg/Lcausing malodors, complaints from the local community and safetyproblems. When the flow from the anaerobe pond then reaches the aerobicpond, energy in the form of aeration providing air causes sulfide to beconverted to sulfate and residual odors associated with the splashing ofthe aerators. If sulfide could be converted to elemental sulfur underanaerobic conditions, then significant costs of aeration could be savedin the region of $90,000 per year, plus the potential for odorcomplaints from the local community would be reduced. The application ofThiosphera pantotropha or purple sulfur bacteria to the inlet of theanaerobe pond could solve this problem by converting the hydrogensulfide to elemental sulfur.

Another application would be in sewer lines or forced lift mains wherehydrogen sulfide accumulates and often has to be vented to costly odortreatment facilities or large amounts of chemicals such as ferricsulfate dosed to precipitate the hydrogen sulfide and prevent itentering the air or where nitrate compounds to be preferentially used bythe SRB's instead of sulfate. In either case, these methods are notaddressing the fundamental biological issue at hand and are extremelyexpensive. An on-site fermentation process could be set-up and used toapply Thiosphera pantotropha or purple sulfur bacteria downstream in thesewer lines to give time for the bacteria to react. Over time apopulation would establish which would cling to the walls and surfacesof objects in the sewer line further enhancing treatment. Otherapplications for hydrogen sulfide include application of Thiospherapantotropha or purple sulfur bacteria to anaerobic digesters, peat bedsto enhance removal or air scrubbers used to strip out hydrogen sulfide.

The fermentation process can be applied to control or displaceundesirable microbes, such as filamentous or Zoogloeal type microbeswhich cause bulking and settleability problems. The fermentation processcan be used to reduce or eliminate secondary polymer and minimize theusage of dewatering chemicals and sludge handling and disposal. Theprocess can be used to enhance removal of organic pollutants in awastewater treatment plant as measured by biological oxygen demand(BOD), chemical oxygen demand (COD), total organic carbon (TOC), ortotal carbon (TC), or specific recalcitrant organic pollutants, such asthose registered as Federal priority pollutants.

The fermentation process can be adapted so that sufficient microbes canbe introduced into a biological wastewater treatment system in order tominimize or avoid the need for the growth phase of the biomass. Byminimizing or avoiding the growth phase of the biomass in the wastewatertreatment system, sludge production can be minimized or eliminated; theneed for supplemental nutrients such as nitrogen and phosphorus can beminimized or eliminated; and the need for oxygen for cell growth, andthe amount of aeration required for treatment can be minimized oreliminated.

The fermentation process can be adapted into a treatment system to treatliquid hazardous waste on-site on a batch basis or on a continuous basisusing microbes specifically isolated to biodegrade the contaminants. Thefermentation process can also be adapted into a treatment system totreat liquid non-hazardous waste using microbes specifically isolated tobiodegrade the contaminants.

Depending of the system being treated and the fermentation process beingused, the fermentation process can be used to turn around a plant on theverge of potential NPDES Permit violation or other environmentalregulation in less than about 5 days; in less than about 4 days; in lessthan about 3 days; in less than about 2 days; or in less than about 1day. Where sufficient microbes are introduced into a biologicalwastewater treatment system, treatment can be achieved almostimmediately.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that the invention may be practiced otherwise than asspecifically described herein.

1-88. (canceled)
 89. A method of reducing the cost required for a wastewater treatment plant for coming into compliance with an environmental discharge regulation comprising: providing an on-site system for growing of microbes at the wastewater treatment plant, the on-site system comprising: a main tank, an input for water, an output for a treatment batch, a mixing apparatus, and a temperature control apparatus; depositing nutrient, water and an inoculum comprising microbes into the on-site system; growing the inoculum in the on-site system to provide a treatment batch comprising an increased number of the microbes; and directly applying at least a portion of the treatment batch, said portion containing microbes, to the polluted wastewater, such that the microbes are not isolated, concentrated or freeze dried between the steps of growing and applying, and wherein the microbes reduce the pollutants in the wastewater as measured by at least one of biochemical oxygen demand, chemical oxygen demand, total organic carbon, or total carbon; wherein the cost required for coming into compliance with the environmental discharge regulation is reduced as compared to the prior cost.
 90. The method of claim 89, wherein the cost required for coming into compliance with the environmental discharge regulation is reduced by at least about 50 percent.
 91. The method of claim 89, wherein the cost required for coming into compliance with the environmental discharge regulation is reduced by at least about 75 percent.
 92. The method of claim 89, wherein the cost required for coming into compliance with the environmental discharge regulation is reduced by at least about 90 percent. 93-95. (canceled) 